System and method for acoustic focusing hardware and implementations

ABSTRACT

The present invention is a method and apparatus for acoustic focusing hardware and implementations.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/213,049, filed Mar. 14, 2014, which is a continuation of U.S. patentapplication Ser. No. 12/209,084 filed Sep. 11, 2008, now U.S. Pat. No.8,714,014, issued May 6, 2014, which application claims priority to andthe benefit of U.S. Provisional Patent Application Ser. No. 61/021,443,entitled “System and Method for Acoustic Focusing Hardware andImplementations”, to Kaduchak, filed on Jan. 16, 2008, and thespecification thereof is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention (Technical Field)

Embodiments of the present invention relate to acoustic cytometry andmore specifically to acoustic focusing hardware and implementations.

BACKGROUND

Note that the following discussion refers to a number of publications byauthor(s) and year of publication, and that due to recent publicationdates certain publications are not to be considered as prior artvis-à-vis the present invention. Discussion of such publications hereinis given for more complete background and is not to be construed as anadmission that such publications are prior art for patentabilitydetermination purposes.

Flow cytometry is a powerful tool used for analysis of particles andcells in a myriad of applications primarily in bioscience research andmedicine. The analytical strength of the technique lies in its abilityto parade single particles (including bioparticles such as cells,bacteria and viruses) through the focused spot of light sources,typically a laser or lasers, in rapid succession, at rates exceedingthousands of particles per second. The high photon flux at this focalspot produces scatter of light by a particle and/or emission of lightfrom the particle or labels attached to the particle that can becollected and analyzed. This gives the user a wealth of informationabout individual particles that can be quickly parleyed into statisticalinformation about populations of particles or cells.

In traditional flow cytometry, particles are flowed through the focusedinterrogation point where a laser directs a laser beam to a focusedpoint that includes the core diameter within the channel. The samplefluid containing particles is hydrodynamically focused to a very smallcore diameter of around 10-50 microns by flowing sheath fluid around thesample stream at a very high volumetric rate on the order of 100-1000times the volumetric rate of the sample. This results in very fastlinear velocities for the focused particles on the order of meters persecond. This in turn means that each particle spends a very limited timein the excitation spot, often only 1-10 microseconds. When the linearflow of the hydrodynamic sheath fluid is stopped, the particles are nolonger focused. Only resuming the hydrodynamic sheath fluid flow willrefocus the particles. Further, once the particle passes theinterrogation point the particle cannot be redirected to theinterrogation point again because the linear flow velocity cannot bereversed. Still further, a particle cannot be held at the interrogationpoint for a user defined period of time for further interrogationbecause focusing is lost without the flow of the hydrodynamic sheathfluid. Because of the very high photon flux at the excitation point,flow cytometry is still a very sensitive technique, but this fasttransit time limits the sensitivity and resolution that can be achieved.Often, greater laser power is used to increase the photon flux in aneffort to extract more signal but this approach is limiting in that toomuch light can often photobleach (or excite to non-radiative states) thefluorophores being used to generate the signal and can increasebackground Rayleigh scatter, Raman scatter and fluorescence as well.

Slower flowing cytometry systems have been developed to push the limitsof sensitivity and have shown detection limits down to the singlemolecule level. In one of these systems, it was shown that lower laserpower (<1 mW) was actually preferable for single molecule detection ofdouble stranded DNA fragments intercalated with fluorescent dyes.Because of the slow transit times (hundreds of microseconds tomilliseconds), it was possible to get maximum fluorescence yield out ofthe dyes while reducing background, photobleaching and non-radiativetriplet states with the lower laser power.

Slow flow hydrodynamic systems, while incredibly sensitive, are not inwidespread use because fluidic dimensions are generally very small,which results in easy clogging and very limited sample throughput. Inorder to focus the sample stream to a core diameter small enough tomaintain the uniform illumination and flow velocity required forprecision particle measurement, the sheath must still be supplied in avery high volumetric ratio to the sample. In order to achieve a slowlinear velocity, the volumetric sample rate must be extremely small.Therefore, to process appreciable numbers of events, the sample must behighly concentrated. If for example a relatively slow linear velocity of1 centimeter per second is desired with a typical core diameter of about10 microns, the sample must be delivered at about 0.05 microliters perminute. To process just 100 cells per second, the cell concentrationmust be 120,000 per microliter or 120 million per milliliter. Thisconcentration requirement in turn makes clogging even more likely. Theproblem is further compounded by the tendency of many types of cells toclump in high concentration and to settle out and stick to surfaces whensample delivery rates are slow. The system created by Doornbos,circumvents the clogging problem by using a conventional flow cell withflow resistors to slow the flow, but he found it very difficult tocontrol precise focused delivery of the sample. This method also doesnot eliminate the need for slow volumetric delivery and highlyconcentrated samples.

Sheathless, non-focusing flow cytometers have been developed but theseinstruments suffer from low sensitivity due to the need for a focal spotsize that will excite particles throughout the channel. The spot size isreduced by using very small capillary channels but particles flow withinthe channel at variable rates according to the laminar flow profile thatdevelops in the channel. This results in different transit times andcoincidence of particles in the laser spot which both make analysis moredifficult. Also, background cannot be reduced by spatially filteringoptics that are designed to collect light from a tightly focused corestream. This limits sensitivity and resolution.

Other approaches have been demonstrated to manipulate particles usingacoustic radiation pressure in a laboratory setting. These devices areplanar devices modeled in Cartesian coordinates. Applying an acousticfield generates a quasi-one-dimensional force field that focusesparticles into a ribbon in a rectangular chamber. For laminar flow, theresulting distribution of particles across the chamber places theparticles in different velocity stream lines. Particles in differentstream lines will not only be in different locations but they will alsoflow at different velocities. This in turn results in differentresidence times for particles at a location within the device. Planarfocusing does not align particles in a manner suitable for use with flowcytometers.

BRIEF SUMMARY OF THE INVENTION

An embodiment of the present invention comprises an acoustic focusingcapillary further comprising a capillary coupled to at least onevibration source and the at least one vibration source possessing agroove. The capillary of this embodiment is preferably coupled to thevibration source at the groove. The groove preferably has anapproximately same cross-sectional geometry as the capillary. Thecapillary can be circular, elliptical, oblate, or rectangular. Thevibration source preferably comprises a piezoelectric material. In thisembodiment, the groove preferably increases an acoustic source apertureof the capillary.

Another embodiment of the present invention comprises a method ofmanufacturing an acoustic focusing capillary. This embodiment furthercomprises providing a capillary and at least one vibration source,machining a groove into the vibration source, and coupling the at leastone vibration source to the capillary at the groove. The groovepreferably has an approximately same cross-sectional geometry as thecapillary. The capillary can be circular, elliptical, oblate, orrectangular. The at least one vibration source preferably comprises apiezoelectric material. This embodiment can optionally compriseincreasing the acoustic source aperture of the capillary.

In yet another embodiment of the present invention, an apparatus thathydrodynamically and acoustically focuses particles in a particle streamcomprises a flow chamber, an outer confine of the flow chamber forflowing a hydrodynamic fluid therethrough, a central core of the flowchamber for flowing the particle sample stream therethrough, and atleast one transducer coupled to the chamber producing acoustic radiationpressure. The transducer of this embodiment is preferably coupled to anouter wall of the flow chamber. The transducer can alternatively form awall of the flow chamber.

A further embodiment of the present invention comprises a method ofhydrodynamically and acoustically focusing a particle stream. Thismethod preferably comprises flowing a sheath fluid into outer confinesof a capillary, flowing a particle stream into a central core of thecapillary, and applying acoustic radiation pressure to the particlestream within the sheath fluid. The particle stream of this method canbe hydrodynamically focused and subsequently acoustically focused.Alternatively, the particle stream is acoustically focused andhydrodynamically focused simultaneously.

Another method of hydrodynamically and acoustically focusing particlesis still a further embodiment of the present invention. This embodimentpreferably comprises providing a fluid comprising particles therein,flowing a sheath fluid into outer confines of a flow chamber, flowingthe fluid containing the particles into a central core of the flowchamber, and applying acoustic radiation pressure to the fluidcomprising the particles. This embodiment can also comprise analyzingthe particles.

One embodiment of the present invention comprises a method of aligningparticles using acoustic radiation pressure. This embodiment preferablyincludes providing a fluid comprising particles therein, subjecting thefluid to acoustic radiation pressure, rotating the fluid 90 degrees, andsubjecting the fluid to acoustic radiation pressure a second time toalign the particles. This embodiment can also comprise analyzing thealigned particles.

Another embodiment of the present invention comprises a method ofhydrodynamically and acoustically focusing particles in a fluid. Thisembodiment includes flowing a fluid comprising particles therein,subjecting the fluid to acoustic radiation pressure in one planardirection to acoustically focus the particles, and flowing a sheathfluid in a second planar direction thereby hydrodynamically focusing thefluid in the second planar direction to further focus the particles.

The present invention further includes methods for dislodging bubbles ina fluidic system. These methods comprise providing a fluid streamthrough a channel and resonating the channel at an acoustic frequency.These methods also include providing a fluid stream through a channeland vibrating the channel walls at a low frequency.

An embodiment of the present invention is an apparatus that acousticallyfocuses particles into a quasi-planar arrangement in a fluid. Thisembodiment preferably comprises a capillary with an oblatecross-sectional geometry and at least one transducer coupled to thecapillary. The capillary is preferably elliptical. This embodiment canfurther comprise an imager for imaging the particles.

Another embodiment of the present invention comprises a method foracoustically focusing particles into a quasi-planar arrangement in afluid comprising particles. The method preferably comprises flowing thefluid comprising particles therein through a flow chamber comprising anoblate cross-sectional geometry and subjecting the fluid to acousticradiation pressure. The cross-sectional geometry of the flow chamber ispreferably elliptical. This embodiment can also include imaging theparticles.

Objects, advantages and novel features, and further scope ofapplicability of the present invention will be set forth in part in thedetailed description to follow, taken in conjunction with theaccompanying drawings, and in part will become apparent to those skilledin the art upon examination of the following, or may be learned bypractice of the invention. The objects and advantages of the inventionmay be realized and attained by means of the instrumentalities andcombinations particularly pointed out in the appended claims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The accompanying drawings, which are incorporated into and form a partof the specification, illustrate one or more embodiments of the presentinvention and, together with the description, serve to explain theprinciples of the invention. The drawings are only for the purpose ofillustrating one or more preferred embodiments of the invention and arenot to be construed as limiting the invention. In the drawings:

FIG. 1 is an embodiment of the present invention illustrating a linedrive capillary where particles are acoustically focused to the centralaxis of the capillary;

FIG. 2 illustrates construction of line-drive capillary with groovedpiezoelectric transducer (PZT) according to one embodiment of thepresent invention;

FIG. 3 illustrates a diagram of a line driven capillary with anelliptical cross section according to one embodiment of the presentinvention;

FIG. 4 illustrate force potential U in a line-driven capillary withelliptical cross section according to one embodiment of the presentinvention;

FIG. 5 illustrate force potential for different aspect ratios for aspherical latex particle in an elliptical cross section, line-drivencapillary according to one embodiment of the present invention;

FIGS. 6A and 6B illustrate focused particle stream flowing through anelliptical cross section line driven capillary according to oneembodiment of the present invention;

FIGS. 7A and 7B illustrates hydrodynamically focused particlesdistributed in a central core stream according to one embodiment of thepresent invention;

FIG. 8 illustrates acoustic focusing of particles in combination withhydrodynamic focusing according to one embodiment of the presentinvention.

FIGS. 9A and 9B illustrates acoustically assisted hydrodynamic focusingaccording to one embodiment of the present invention; and

FIG. 10 illustrates a combination of acoustic and hydrodynamic focusingin a microfluidic channel according to one embodiment of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

As used herein “a” means one or more.

As used herein “flow chamber” means a channel or capillary having ashape selected from rectangular, square, elliptical, oblate circular,round, octagonal, heptagonal, hexagonal, pentagonal, and triagonal. Itis not necessary for the shape of the interior walls of the flow chamberbe the same as the shape of the exterior walls. As a non-limitingexample, a flow chamber can have an interior wall defined by a circularshape and the exterior wall defined by a rectangular shape.Additionally, the flow chamber can be part of a composite constructionof materials and geometries wherein one of the above shapes defines theinterior shape of the flow chamber.

As used herein “capillary” means a channel or chamber having a shapeselected from rectangular, square, elliptical, oblate circular, round,octagonal, heptagonal, hexagonal, pentagonal, and triagonal. It is notnecessary for the shape of the interior walls of the capillary be thesame as the shape of the exterior walls. As a non-limiting example, acapillary can have an interior wall defined by a circular shape and theexterior wall defined by a rectangular shape.

One aspect of one embodiment of the present invention provides for easein alignment during device fabrication and for larger acoustic sourceapertures. Another aspect of one embodiment of the present inventionprovides for a line-driven capillary with oblate cross-section to obtainquasi-planar particle concentration. Another aspect provides for planarparticle concentration without particles contacting and/or staying incontact with the inner capillary wall. Another aspect provides forimaging applications where particles spread over a plane in a narrowdepth of field. Another aspect provides for applying acoustic radiationpressure forces to assist in stabilizing standard hydrodynamic particlefocusing systems. Yet another aspect provides for reduced sheathconsumption in slow-flow hydrodynamic systems and to assist in particlefocusing in planar systems (e.g. chip based systems). Still anotheraspect provides a method to dislodge bubbles from fluidic systems.

Construction of Line-Driven Capillaries w/Grooved Source

Line-driven capillaries are used to acoustically concentrate particlesin a flowing stream within a capillary. Particles experience atime-averaged acoustic force resulting from acoustic radiation pressure.FIG. 1 illustrates line-driven capillary 10 operating in a dipole modewhere particles 12 are acoustically focused to the central axis ofcapillary 14 to the position of an acoustically formed particle trapaccording to one embodiment of the present invention. (The embodimentillustrated in FIG. 1 is applicable to any vibrational mode of thesystem whether it be monopole, dipole, quadrapole, etc., or acombination of modes.) It is possible to drive different modeconfigurations with different spatial configurations of sources attachedto the capillary.

Another aspect of one embodiment of the present invention provides aline-driven capillary system that both delivers stable acoustic signalswithin the capillary and possess consistent, repeatable electricalproperties of the electromechanical circuit that drives the system.

In one embodiment of the present invention, line-driven capillary 10 iscomprised of capillary 14 coupled to vibration source 16. Capillary 14can be made from, but are not limited to glass, metal, plastic, or anycombination thereof. Low loss materials are preferably particleconcentrators and stainless steel is one of the better capillarymaterials. Vibration source 16 is preferably comprised of apiezoelectric material. Examples of piezoelectric materials include butare not limited to PZT, lithium niobate, quartz, and combinationsthereof. Vibration source 16 can also be a vibration generator such as aLangevin transducer or any other material or device that is capable ofgenerating a vibration or surface displacement of the capillary. Anotheraspect of the embodiment of the present invention comprises anacoustically focused line drive capillary that yields a larger acousticsource aperture than a standard line contact.

According to one embodiment of the present invention, groove 18 ismachined into vibration source 16 into which capillary 14 is cradled, asillustrated in FIG. 2. A diagram is shown in FIG. 2, which comprisesline-drive capillary 10 with grooved vibration source 16, a small PZTslab with machined circular groove 18 is adhered to capillary 14 toimprove manufacturability and acoustic performance. Groove 18 iscircular with a radius that matches the outer radius of capillary 14plus a small glue layer. The number of grooved vibration sourcesattached to capillary 14 is not limited to one. Using more than onegrooved vibration source is advantageous in driving different acousticmodes that require specific spatial dependence. For example, a dipolemode is driven with a single source or with two sources attached toopposite walls of capillary 14 and driven 180 degrees out of phase. Aquadrapole mode is driven by attaching sources at orthogonal positions(90 degree offset from one another) and driven out of phase. Forcapillaries of non-circular cross section, groove 18 will typically takeon the cross sectional geometry of capillary 14. For example, anelliptical cross section capillary would require an elliptical crosssection groove. Capillary 14 is preferably held to vibration source 16with a small glue layer. When using a piezoelectric crystal as vibrationsource 16, it is not necessary to have an electrical conducting layerinside groove 18 that is cut into the crystal. Construction with andwithout conductors in groove 18 have been demonstrated.

Another aspect of one embodiment of the present invention provides easeof device construction.

Yet another aspect of one embodiment of the present invention providesfor larger acoustic source aperture as compared to a true line-drivendevice.

Still another aspect of one embodiment of the present invention providesfor repeatable acoustic/electrical performance.

Another aspect of one embodiment of the present invention provides forease in alignment of capillary 14 with vibration source 16.

Still another aspect of one embodiment of the present invention providesfor a larger glue surface upon which to attach a transducer.

Additionally, it is not necessary for the capillary to have a circularcross section. In one embodiment of the present invention, a squarecross section groove in PZT is used. Capillaries can be constructed withmany geometries including but not limited to elliptical, square,rectangular, general oblate shapes, as well as any cross sectionalgeometry.

Quasi-Planar Focusing of Particles in Line-Driven Oblate Capillaries

Referring now to FIG. 3, line-driven capillaries with circular crosssections can be driven to align particles along the axis of thecylindrical capillary when driven in a dipole mode in one embodiment ofthe present invention. In this embodiment, it may be desirable incertain applications to localize the particles only to a specific planewithin a capillary, not to a point or line. This is the case for imagingapplications where particles need to be distributed in a plane within anarrow depth of field of the imaging optics. A method to spatiallydistribute the particles is to break the circular symmetry of thesystem. By making the cross section of the capillary more oblate (e.g.elliptical), it is possible to keep tight spatial localization in onedimension while allowing the particles to be spatially distributed inanother dimension. This method is advantageous for systems requiringparticles placed in a planar (or quasi-planar) arrangement.

For example, an acoustically driven capillary with an elliptical crosssection is illustrated in FIG. 3. In this embodiment, acoustic forcespatially distributes particles in a plane along the major axis andtightly confines particles along the minor axis. The aspect ratio A ofthe ellipse is given by the ratio of the minor axis ay to major axis ax:A=ay/ax. To calculate the acoustic force on particles within thecapillary, the acoustic radiation pressure force on a compressible,spherical particle of volume Vin an arbitrary acoustic field can bewritten in terms of an acoustic radiation pressure force potential U(Gor'kov 1962):

$U = {\frac{4}{3}\pi \; {a^{2}\left\lbrack {{\left( {\beta_{o}\frac{\left( p^{2} \right)}{2}} \right)f_{1}} - {\frac{3}{2}\left( \frac{p_{o}\left( v^{2} \right)}{2} \right)f_{2}}} \right\rbrack}}$

Here, a is the particle radius, β₀ is the compressibility of thesurrounding fluid, and ρ₀ is the density of the surrounding fluid. Thepressure and velocity of the acoustic field in the absence of theparticle are described by p and v, respectively, and the bracketscorrespond to a time-averaged quantity. The terms f₁ and f₂ are thecontrast terms that determine how the mechanical properties of theparticle differ from the background medium. They are given by:

$f_{1} = {1 - \frac{\beta_{p}}{\beta_{o}}}$$f_{2} = \frac{2\left( {\rho_{p} - \rho_{o}} \right)}{\left( {{2\rho_{p}} - \rho_{o}} \right)}$

The subscript p corresponds to intrinsic properties of the particle. Theforce F acting on a particle is related to the gradient of the forcepotential by:

F=−vU

Particles are localized at positions where the potential U displays aminimum. (For a circular cross section capillary, a potential minimum iscoincident with the axis of the capillary forming the particle trap inFIG. 1.)

The force potential U for an elliptical cross section capillaryline-driven in a dipole type mode is illustrated in FIG. 4. Thepotential is calculated for latex spheres in water. In thisconfiguration, the particles experience a force that transports them toa potential well that appears to stretch between the foci of theellipse. The particles are also more tightly focused in the direction ofthe minor axis and are more “spread out” in the direction of the majoraxis.

Depending upon the aspect ratio of the ellipse, the force potential inorthogonal directions can be dramatically different. This is illustratedin FIG. 5. In FIG. 5, force potential for different aspect ratios for aspherical latex particle in an elliptical cross section, line drivencapillary is shown. For this configuration, the particles are morelocalized along the minor axis and less localized along the major axis.A change in frequency can cause greater localization along the majoraxis and less localization along the minor axis. For aspect ratioscloser to unity, the potential well is more pronounced than for aspectratios further from unity. Note the gradient of the potential is smallerin the direction of the major axis. The reduced gradient implies lesslocalization of the particles along this direction. As the aspect ratioof the ellipse decreases, the potential well depth decreases resultingin milder gradients and less localization. Therefore, with decreasingaspect ratio, particles experience a greater spread along the major axisof the ellipse (reduced force due to reduced gradient). (There is also aspreading of particles along the minor axis, but to much less of anextent than in the direction of the major axis.)

Results showing this effect are given in FIG. 6. FIG. 6(a) displays anexample of particles flowing through an elliptic cross sectioncapillary. In this example, the particles are approximately 5.6 mmdiameter fluorescent latex spheres (more specifically, polystyrenebeads) and appear as horizontal streaks in the image. Flow is from leftto right. The image plane contains the major axis of the ellipse and thecentral axis of the capillary. The particles are spread acrossapproximately half the width of the capillary forming a ribbon ofparticles. In this example, there is enough force on the particlesdirected toward the axis of the capillary to keep them off the walls. InFIG. 6(b), the image plane has been rotated 90 degrees to include theminor axis of the ellipse and the central axis of the capillary. In thisdirection, the gradient along the potential well is greater leading to agreater confinement of the particles along the capillary axis. Here theyare confined to a single line coincident with the central axis of thecapillary.

Several characteristics of these types of modes exist:

Particles can be tightly focused either along major or minor axis ofellipse with ‘loose’ focusing along the orthogonal direction (selectionof which axis is the weak focusing direction is mode dependent).

In the weakly focused dimension, enough force can exist to keepparticles off the wall of the device.

Particles are confined to a plane which is conducive to imagingapplications where it is necessary to place particles in a common planeespecially when depth of focus is small.

Acoustically Assisted Hydrodynamic Focusing of Particles

Hydrodynamically-focused particle streams are used in flow cytometry aswell as other areas where precisely aligned particles in a flowing corestream are required. Hydrodynamic focusing is traditionally employed inflow cytometry to focus particles into a tight stream for laserinterrogation. A diagram of a hydrodynamically focused particle streamis illustrated in FIG. 7(a). In this example, a sample is injected intoa central core stream contained within a coaxial sheath flow. The sheathfluid is typically a clean buffer solution traveling at many times thevelocity of the sample input in order to hydrodynamically confine thecentral sample stream into a smaller cross sectional area. This actionconfines the particles in a cylindrical core stream of very narrowwidth. The hydrodynamically focused core stream radius r is givenapproximately as

$r = \sqrt{\frac{Q}{\pi \; v}}$

where Q is the volumetric flow rate of the core stream and v is thevelocity of the core stream. Note that larger volumetric sample deliveryand/or lower velocities yield larger diameters of the core stream.

Hydrodynamically focused sample streams can suffer from instabilities ofthe central core stream position as a function of many factors. Thesecan include but are not limited to nucleation of bubbles on the cellwalls that alter stream lines, turbulence, and combinations thereof. Itis advantageous to assist hydrodynamically focused systems with anexternal force that stabilizes the spatial position of the central corestream. An embodiment of the present invention comprises a device thatuses multiple fluidic streams to steer the central core stream.

FIG. 7(b) illustrates an embodiment of the present invention comprisingan acoustically assisted hydrodynamically focused sample stream. In FIG.7(b), an outer coaxial flow of sheath fluid confines the central corestream containing the sample. By applying acoustic forces to theparticles in the hydrodynamically focused core stream, the particles arepreferably focused further within the stream.

One embodiment of the present invention combines acoustic focusing ofparticles with hydrodynamic focusing. Acoustic focusing assistshydrodynamic focusing systems by stabilizing the absolute location ofthe particle stream against external forces. Acoustic focusing is alsoused to further tighten the focus of the particle stream within ahydrodynamically focused system where reduction in sheath fluidconsumption or increase in sample throughput is desired without the lossof particle focus quality within the stream. This is particularlyimportant for applications where the sample is dilute. A prime exampleis high speed sorting of “sticky” cells that must be kept at lowerconcentration to prevent aggregation. Another example is where reductionof sheath fluid is a priority without sacrificing particle focus.Furthermore, some systems that employ acoustic focusing may not want thesample to contact the walls. (For example, this will keep the build-upof proteins and small particles that are unaffected by acousticradiation pressure off the capillary walls. These systems can use aslow, low-volume sheath to entrain the sample. Acoustic focusing canthen be used to tightly focus the particles within the sample stream.)

An example of acoustically assisted hydrodynamic focusing is shown inFIG. 7(b). In this example, a standard hydrodynamically-focused systemis outfitted with ultrasonic transducers to set up a standing wave inthe fluid cavity. The particles are initially hydrodynamically focused.Ultrasonic radiation pressure then forces the particles to a forcepotential minimum located along the axis of the central core streamwhere they are further aligned within the central core stream.

Referring now to FIG. 8, a schematic of a device capable of applyingacoustic focusing prior to, during, or both prior to and duringhydrodynamic focus as illustrated according to one embodiment of thepresent invention. This embodiment comprises sample 20 flowing throughcapillary 22. Sheath fluid 24 hydrodynamically focusing particles 26.Transducers 28 and 30 acoustically focus particles 26 along the axis ofthe central core prior to hydrodynamic focusing while transducers 32 and34 acoustically focus particles 26 during hydrodynamically focusing.

Measurements demonstrating acoustically assisted hydrodynamic focusingare illustrated in FIG. 9. The image on the left demonstrateshydrodynamic focusing in a cylindrical channel of width 500 microns. Thecentral core stream comprises approximately 5.6 μm diameter polystyreneparticles in solution (approx. 0.0025% by volume). The central corestream is surrounded by a coaxial sheath flow that contains a phosphatebuffer solution. The image on the left shows particles in the centralcore stream during hydrodynamic focusing. The sheath fluid is introducedat a volumetric flow rate of between approximately 100 to 1,000microL/min and preferably at a rate of approximately 400 microL/min, andthe sample core stream is introduced at a volumetric flow rate ofbetween 50 to 500 microL/min and preferably at a rate of approximately100 microL/min. The frequency of the acoustic excitation is 2.1 MHz. Inthe image on the right, an acoustic field is activated that is designedto produce a particle trap along the axis of the core sample stream.Particles within the core sample stream are further isolated within thecore stream by the acoustic field.

Aspects of acoustically assisting hydrodynamically focused samplestreams include but are not limited to:

-   -   Repeatable location of focused particle stream    -   Increased particle focusing in lower velocity hydrodynamically        focused streams thereby reducing the sheath fluid requirements        (particles spatially confined to a stream smaller in diameter        than core stream)    -   Increased sample throughput of dilute samples while maintaining        tight spatial positioning    -   Less effect of turbulence and other exterior influences on the        exact location of the focused particles    -   Method to isolate sample stream from capillary walls in a system        where predominant particle focusing is conducted by acoustic        radiation pressure (e.g. line-driven capillary)

There are many different arrangements where both acoustically focusingand hydrodynamically focusing can be advantageous. FIG. 7(b) displays adevice with two transducers attached to a hydrodynamic focusing cell.The acoustic field can be used in a cell that is circular, square, orany other geometry. The number of transducers shown in FIG. 7(b) is two.The minimum number of transducers is one. Using more than one transducerprovides feedback to monitor the acoustic field within the chamber.Additionally transducers may be attached in orthogonal directions tocreate force fields that are optimized for a given application. In anembodiment of the present invention, it is advantageous to focus theparticles single file into a line within the core sample stream. Inanother embodiment of the present invention, it is advantageous to focusparticles in one dimension and allow them to spread out in an orthogonaldirection.

The ability to use acoustically assisted hydrodynamic focusing ofparticles is also advantageous for applications in microfluidics.Hydrodynamic focused particle streams in microchannels, microfluidicchips, or other rectangular (or quasi-rectangular) channel geometricscan be enhanced by combining acoustic focusing and hydrodynamicfocusing. In one embodiment of the present invention and to reducesheath fluid consumption, one dimensional hydrodynamic focusing can beused, as illustrated in FIG. 10. A combination acoustic and hydrodynamicfocusing in a microfluidic channel is shown. Hydrodynamic focusinglocalizes particles in the horizontal direction and acoustic focusinglocalizes particles in the vertical direction. Ease of implementationfor both acoustic and hydrodynamic focusing in planar devices isaccomplished in this embodiment. (This can also work if the forcediagram in FIG. 10 is rotated 90 degrees.) Using both acoustic focusingand hydrodynamic focusing also keeps small particles and molecularspecies from contacting the channel walls.

Another embodiment of the present invention allows for serial acousticfocusing in microfluidic applications. Acoustic focusing applied to aflowing stream of particles in a quasi-rectangular cross section chamberfocuses particles into a ribbon-like structure. In order to preserve thelayered construction used in many microfluidic assemblies, it isadvantageous to preserve the placement of the transducers in parallelplanes. Thus, a method to focus particles into a narrow spatialconfiguration involves acoustically focusing particles into a plane,rotating the flow by 90 degrees, and then acoustically focusing againinto the new orthogonal plane. The net result is a narrow spatialdistribution of particles. When the transducer is used to excite adipole type mode within the flow chamber, the result is particlesnarrowly focused about the central axis of the flow chamber.

Bubble Dislodging from Fluidic Systems

In traditional flow cytometry, bubbles that adhere to walls of a fluidicsystem are problematic. They can interfere by moving laminar flow lines,affecting local reactions, deviating focused particle streams. Forexample, in flow cytometers, bubbles in a fluidic system can have theeffect of moving the position of the hydrodynamically-focused samplestream. This movement appears as a misalignment of the optical system tothe user and a recalibration is required. A technique to dislodgebubbles from nucleation sites in fluidic systems, especiallymicrofluidic systems, is very desirable.

Acoustic radiation pressure has been shown to have a large effect onbubbles in fluids due to the large mismatch in density andcompressibility between liquids and gases. Acoustic energy can be usedto dislodge bubbles from fluidic system in several different ways.

In one embodiment of the present invention, a fluidic system isengineered such that when resonated at an appropriate acousticfrequency, bubbles experience a force that pulls them away from the walland stabilizes their equilibrium position within the fluid stream thatexists at the location of a pressure node (for bubbles driven atfrequencies below their monopole resonance). The chamber is preferablydriven acoustically with either an internal acoustic source ornoninvasively with a source attached to an outer wall of the chamber.This is a robust method to dislodge bubbles from walls.

In another embodiment of the present invention, vibration of the channelwalls at low frequencies is preferably used to dislodge the bubbles. Byvibrating the wall as part of a structural resonance of the system,large surface displacements are achieved. (These displacements aretypically larger at lower frequencies.) Large forces coupled with largedisplacements are preferably used to break the bond between the bubbleand the chamber surface. The inertial forces coupled with localizedfluid flows at the chamber wall surface are effective at bubbledislodgement.

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

Although the invention has been described in detail with particularreference to these preferred embodiments, other embodiments can achievethe same results. Variations and modifications of the present inventionwill be obvious to those skilled in the art and it is intended to coverin the appended claims all such modifications and equivalents. Theentire disclosures of all references, applications, patents, andpublications cited above and/or in the attachments, and of thecorresponding application(s) are hereby incorporated by reference.

1-36. (canceled)
 37. A method for focusing particles, comprising: to afluid comprising particles, the fluid being disposed within a flowchamber having an oblate cross section that has a minor axis and a majoraxis, applying acoustic radiation pressure from a vibration source suchthat the particles are acoustically focused in a plane along the majoraxis and at a location along the minor axis.
 38. The method of claim 37,wherein the fluid is flowing within the flow chamber.
 39. The method ofclaim 37, wherein the oblate cross section of the flow chamber ischaracterized as elliptical.
 40. The method of claim 37, wherein theoblate cross section of the flow chamber is characterized asrectangular.
 41. The method of claim 37, wherein the vibration sourcecomprises a groove to which the flow chamber is coupled.
 42. The methodof claim 37, further comprising applying a hydrodynamic force to theparticles.
 43. The method of claim 42, wherein the acoustic radiationpressure is applied before the application of the hydrodynamic force.44. The method of claim 42, wherein the acoustic radiation pressure isapplied after the application of the hydrodynamic force.
 45. The methodof claim 37, further comprising imaging the particles.
 46. A method ofhydrodynamically and acoustically focusing a particle stream,comprising: flowing a sheath fluid into outer confines of a flowchamber; flowing a particle stream into a central core of the flowchamber; and applying acoustic radiation pressure to the particlestream.
 47. The method of claim 46, wherein (a) at least some of theparticles of the particle stream are hydrodynamically focused by thesheath fluid and then acoustically focused by the acoustic radiationpressure, or (b) at least some of the particles of the particle streamare acoustically focused by the acoustic radiation pressure and thenhydrodynamically focused by the sheath fluid.
 48. The method of claim46, wherein the particles of the particle stream are acousticallyfocused by the acoustic radiation pressure while within the sheathfluid.
 49. The method of claim 46, further comprising imaging theparticles.
 50. The method of claim 46, wherein the flow chamber has anoblate cross section.
 51. The method of claim 46, wherein the acousticradiation pressure is applied in one planar direction to acousticallyfocus the particles and the sheath fluid is flowed in a second planardirection so as to further focus the particles.
 52. A system,comprising: a flow chamber, an outer confine of said flow chamber havingan inlet for flowing a sheath fluid therethrough; a central core of saidflow chamber for flowing a particle sample stream therethrough; and atleast one transducer coupled to said chamber producing acousticradiation pressure.
 53. The system of claim 52, wherein the flow chamberhas an oblate cross section.
 54. The system of claim 52, furthercomprising an imager configured to image particles of the particlesample stream.
 55. The system of claim 52, wherein the flow chamberdefines a fluid pathway, and wherein the at least one transducer isdownstream of the sheath fluid inlet along the fluid pathway.
 56. Thesystem of claim 52, wherein the flow chamber defines a fluid pathway,and wherein the at least one transducer is upstream of the inlet alongthe fluid pathway.